R.J. Siezen

Complete Genome Sequence of Lactococcus lactis subsp. lactis KF147, a Plant-Associated Lactic Acid Bacterium

Lactococcus lactis is a lactic acid bacterium used in the production of many fermented dairy products. We report the complete genome sequence of L. lactis subsp. lactis KF147, a nondairy strain isolated from mung bean sprouts. The circular chromosome of 2,598,144 bp, the largest among the sequenced lactococcal strains, encodes many properties related to adaptation to the plant environment.

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome.

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.

Comparative genomics of Lactobacillus.

The genus Lactobacillus includes a diverse group of bacteria consisting of many species that are associated with fermentations of plants, meat or milk. In addition, various lactobacilli are natural inhabitants of the intestinal tract of humans and other animals. Finally, several Lactobacillus strains are marketed as probiotics as their consumption can confer a health benefit to host. Presently, 154 Lactobacillus species are known and a growing fraction of these are subject to draft genome sequencing. However, complete genome sequences are needed to provide a platform for detailed genomic comparisons. Therefore, we selected a total of 20 genomes of various Lactobacillus strains for which complete genomic sequences have been reported. These genomes had sizes varying from 1.8 to 3.3u2003Mb and other characteristic features, such as G+C content that ranged from 33% to 51%. The Lactobacillus pan genome was found to consist of approximately 14u2003000 protein‐encoding genes while all 20 genomes shared a total of 383 sets of orthologous genes that defined the Lactobacillus core genome (LCG). Based on advanced phylogeny of the proteins encoded by this LCG, we grouped the 20 strains into three main groups and defined core group genes present in all genomes of a single group, signature group genes shared in all genomes of one group but absent in all other Lactobacillus genomes, and Group‐specific ORFans present in core group genes of one group and absent in all other complete genomes. The latter are of specific value in defining the different groups of genomes. The study provides a platform for present individual comparisons as well as future analysis of new Lactobacillus genomes.

Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95°C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including αS1-casein and synthetic peptides.

Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans

Abstract. The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.

The human gut microbiome: are we our enterotypes?

Blood groups, finger prints, iris scans and DNA bar codes are several ways to distinguish or identify individual humans. According to Arumugam and colleagues (2011) we can now also be distinguished by the microbial communities in our faeces, our ‘enterotypes’. n nThe human gut is one of the most densely populated ecosystems known, and although this ecosystem contains members of the three domains of life – bacteria, archaea and eucarya (Finegold etu2003al., 1983) – it is dominated by bacteria. They are essential in digesting the food we eat and in keeping us healthy by stimulating our immune system and fighting off pathogenic bacteria. It is estimated that 1013–1014 microbes inhabit our gastrointestinal tract (GIT), with the greatest number residing in the distal gut, where they synthesize essential vitamins and process otherwise indigestible components of our diet such as plant polysaccharides (Backhed etu2003al., 2005). Early studies used 16S ribosomal DNA (rDNA) analysis to type and enumerate the distal gut and faecal microbiota. More than 90% of the bacterial phylotypes present in the intestinal microbiota in healthy humans are members of only three bacterial divisions: the Bacteroidetes, the Firmicutes and the Actinobacteria (Zoetendal etu2003al., 2006). n nNevertheless, each healthy adults gut appears to have a unique and relatively stable microbiota (Zoetendal etu2003al., 1998; Turnbaugh etu2003al., 2007), which is a reflection of the numerous different phylogenetic clusters among the Firmicutes, Clostridium clusters IV, IX and XIVa, including the predominant genera Clostridium, Eubacterium, Roseburia and Ruminococcus. Furthermore, the Actinobacteria that encompass mainly the genera Bifidobacterium and Atopobium also represent important members of the gut microbial community (Harmsen etu2003al., 2002; Turroni etu2003al., 2008). Notably, recent estimates of the diversity of the human gut microbial ecosystem indicate it may encompass more than 1000 species and a multitude of strains (Backhed etu2003al., 2005; Blaut and Clavel, 2007; Rajilic‐Stojanovic etu2003al., 2007). Microbiota composition studies in humans have discovered that aberrations in the microbiome composition is present in obese individuals (Ley etu2003al., 2006; Turnbaugh etu2003al., 2009) as well as in individuals with a variety of other diseases (Zoetendal etu2003al., 2008). n nSince the largest part (∼80%) of gut microbes remains uncultured, metagenomics analysis has become fashionable in the past 5 years to estimate the types, relative abundance and genome content of microbes in various parts of the GIT. The first metagenomics analysis from just two faecal samples (Gill etu2003al., 2006) led to early insight into enrichment of genes encoding specific metabolic pathways, including metabolism associated with glycans, amino acids and xenobiotics, but also methanogenesis and biosynthesis of vitamins and isoprenoids. However, this early study provided only a very fragmented view due to limitations in sample size, sequencing technology, number of bases sequenced and availability of suitable reference genomes of gut inhabitants.

Protein Engineering and Biosynthesis of Nisin and Regulation of Transcription of the Structural nisA Gene

Abstract The lantibiotic nisin, produced by Lactococcus lactis , is an antimicrobial peptide characterized by the presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (β-methyl)lanthionine rings, which are formed post-translationally. Nisin is widely used in the food industry as a preservative, since it inhibits the growth of unwanted gram-positive bacteria. One of the objectives of our research is to get insight in the complex biosynthesis and regulation of production of nisin. The structure and function of several biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, biosynthesis, secretion and immunity. An exciting finding is that nisin itself, when added to the culture medium, can induce the transcription of its own structural gene. Another goal is to design and produce altered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stability, solubility or activity, recent results on the protein engineering of residues Ile1, Dhb2, AlaS3, Lys12, AbuS13, Met17, Asn20 and Met21 indicate that (i) residue 1 can be replaced without dramatic loss of activity; (ii) the presence of a Thr residue at position 2 significantly lowers the antimicrobial potency, whereas the presence of a Dha residue at position 2 improves activity; (iii) the replacement ofAlaS3 by AbuS leads to a dramatic loss of activity, probably due to a conformational change in the first lanthionine ring; (iv) the integrity and hydrophobicity of ring 3 are important for antimicrobial activity; and (v) the hinge region between rings 3 and 4 is important but not essential for antimicrobial activity.

Purification, characterization, and molecular modeling of pyrolysin and other extracellular thermostable serine proteases from hyperthermophilic microorganisms.

Publisher Summary Currently several serine proteases have been purified from hyperthermophiles with a variety of approaches and characterized at the enzyme and, in most cases also the gene level. Those that are genetically characterized all belong to the subtilisin-like family of serine proteases, also known as “subtilases.” Moreover, a variety of homologs of subtilases can be detected in the present databases of sequenced genomes of hyperthermophiles. The first of these serine proteases to be characterized at the enzyme and gene level has been pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus , which is the most thermostable protease to date and retains half of its activity after boiling for several hours. A characteristic feature of pyrolysin and other proteases is the fact that these enzymes are substrates of their proteolytic activity and degrade themselves in a process termed autoproteolysis. This has for a long time hampered the purification of pyrolysin and prevented its further characterization at the molecular level. This chapter discusses the procedures for the biochemical and genetic characterization of pyrolysin and other related thermostable serine proteases and the prediction of their properties by homology comparisons, database searches, and molecular modelling.

PanCGHweb: a web tool for genotype calling in pangenome CGH data.

Summary: A pangenome is the total of genes present in strains of the same species. Pangenome microarrays allow determining the genomic content of bacterial strains more accurately than conventional comparative genome hybridization microarrays. PanCGHweb is the first tool that effectively calls genotype based on pangenome microarray data. Availability: PanCGHweb, the web tool is accessible from: http://bamics2.cmbi.ru.nl/websoftware/pancgh/ Contact: sacha.vanhijum@nizo.nl

Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme.

ABSTRACT The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.